Resorbable collagen membrane for use in guided tissue regeneration

ABSTRACT

The invention is concerned with wound healing and in particular with the use of a collagen-containing membrane in guided tissue regeneration. The invention provides a resorbable collagen membrane for use in guided tissue regeneration wherein one face of the membrane is fibrous thereby allowing cell growth thereon and the opposite face of the membrane is smooth, thereby inhibiting cell adhesion thereon.

The present invention is concerned with wound healing and in particularwith the use of a collagen-containing membrane in guided tissueregeneration.

In any situation, such as following surgery especially oral or dentalsurgery, where wound healing is desirable, it has proved to be difficultto provide conditions which prevent ingrowth of other tissues into thearea where regeneration is required. Thus, for example, where asubstantial portion of a tooth root is removed due to decay or disease,it is desirable that healthy bone regeneration occurs to replace thebone tissue removed. However, it has been found that the cavity left byremoval of the bone is quickly filled by connective tissue and that thisingrowth of connective tissue effectively prevents bone regeneration.

In order to overcome such difficulties the technique known as "guidedtissue regeneration" has been developed. In this method a membrane issurgically inserted around the periphery of the wound cavity. Themembrane prevents or hinders the invasion of the wound cavity byunwanted cell types and thus allows the preferred cells to grow into thecavity, thereby healing the wound.

Two membrane types are currently used in guided tissue regeneration:

1) Synthetic, non-resorbable PTFE membranes, such as Goretex (trademark); and

2) Synthetic resorbable membranes formed from glycolide and lactidecopolymers.

However, both of these membrane types suffer from serious disadvantages.The PTFE membrane, although exhibiting suitable characteristics ofporosity, strength and flexibility, remains non-resorbable and thereforea second surgical operation is required to remove the membrane. Therequirement for further surgical procedures may be traumatic for thepatient and may also damage the new tissue regenerated thus extendingthe treatment period.

The second membrane type is woven from glycolide and lactide copolymerfibres. Whilst this membrane is resorbable the breakdown products areirritant and this irritance may have undesirable effects on the patient.

Both prior art membranes act as filters, allowing liquids to pass freelyand forming a barrier to cells. However, the membrane surface is not"cell-friendly", ie. it does not stabilise blood clots or support cellgrowth. Consequently, neither of the prior art membranes provide optimalconditions for cell growth and wound healing.

We have now found a membrane with ideal characteristics for guidedtissue regeneration.

The present invention provides a resorbable collagen membrane for use inguided tissue regeneration wherein one face of said membrane is fibrousthereby allowing cell growth thereon and the opposite face of saidmembrane is smooth, thereby inhibiting cell adhesion thereon.

The two opposing sides or faces of the membrane thus have differenttextures which affect cell growth in different ways.

The smooth side acts as a barrier or filter to hinder cell ingrowth andwill prevent undesirable cell types from colonising the cavity describedby the membrane through physical separation. By contrast, the fibrousside of the membrane is haemostatic (stabilises blood clots) and aidscell growth by providing a suitable support for the new cells. In use,therefore, the membrane should be inserted with the smooth sideoutermost and the fibrous side facing the cells where regeneration isdesired.

The membrane for use in guided tissue regeneration according to thepresent invention may be derived from a natural collagen membrane. Beingderived from a natural source, the membrane for use in the presentinvention is totally resorbable in the body and does not form toxicdegradation products.

Further the membrane has a tear strength and tear propagation resistancecomparable to that of textile material in both wet and dry states. Themembrane can therefore be surgically stitched if required. The membranematerial is strongly hydrophilic and has good adherence when wetallowing several layers to be stacked together. When moist the materialis very elastic which allows irregularly shaped or uneven wounds to beproperly covered.

In both humans and animals, certain membranes surrounding importantorgans and separating different tissues and cells are made up ofcollagen. Examples of such membranes include the pericardium andplacental membranes on the macroscale and basal membranes on themicroscale.

Collagen products are now used widely in medicine. A variety of collagenmaterials are available including soluble collagen, collagen fibres,sponges, membranes and bone implants allowing diverse usage of thismaterial, for example, collagen fibres and sponges for haemostasis,collagen membranes for wound covering or implantation, and injections ofsoluble collagen in plastic surgery. Nonetheless, it has not previouslybeen recognised that a collagen membrane would be suitable for guidedtissue regeneration.

Various artificial collagen-containing membranes have been described inthe prior cut and proposed for the dressing or coverage of wounds. Thus,in WO-A-88/08305 (The Regents of The University of California) there isdescribed a composite skin replacement which consists of a layer ofhuman epidermal cells together with a layer of a biosynthetic membranewhich may be formed of collagen and mucopolysaccharides. However, thecollagen/mucopolysaccharide portion is of uniform texture throughout anddoes not exhibit the properties of the membrane proposed herein forguided tissue regeneration. Furthermore, such membranes are stronglyimmuno-reactive and can only be used on the donor of the cells. Anotherartificial collagen-containing membrane is described in DE-A-2631909(Massachusetts Institute of Technology). This membrane consists of aminimum of two layers, the first layer being a combination of collagenand mucopolysaccharides and the second layer being a synthetic polymersuch as a polyacrylate. However, this membrane is totallynon-resorbable, the collagenous layer being so tightly cross-linkedinternally that resorption cannot occur.

The membrane for use in the present invention may be derived directlyfrom naturally occurring membranes which, as far as possible, retaintheir natural collagen structure. The membrane sources include sectionsof hide with a grain side, tendons, various animal membranes etc. Apreferred source of membrane is the naturally occurring peritoneummembrane, especially taken from calves or piglets. Peritoneum membranesfrom young pigs aged 6-7 weeks old (weighing 60-80 kg) are especiallypreferred.

The membrane material for use in the present invention should preferablyconsist of pure, native (not denatured), insoluble collagen. However, inan animal's body, collagen is accompanied by a number of substanceswhich have undesirable chemical, physical and/or physiologicalproperties. The collagen therefore has to be freed from these substancesby purification. Since the nature of such substances variesconsiderably, enzymatic purification is virtually impossible. It is thuspreferable to carry out purification chemically, taking care to minimiseany alteration to the chemical structure of the collagen and thus tomaintain its original native properties.

According to a further aspect of the present invention we provide amethod of preparing a membrane as described above in which a mammaliancollagen membrane having a smooth face and a fibrous face is subjectedto treatment with alkali to saponify fats and degrade alkali sensitivesubstances and then acidified to degrade acid sensitive substances,followed by washing, drying, degreasing and optional cross-linking.

During purification, the following changes occur:

non-collagenous proteins are eliminated

glycosaminoglycans and proteoglycans are dissolved and eliminated

the fats are partially saponified, and totally eliminated.

During such treatment, the following undesirable changes may also occur:

hydrolysis of the amide groups of asparagine and glutamine

a shift in the isoelectric point

cleavage of crosslinking bonds

transamidation, with the formation of isopeptides

racemisation of amino acids

cleavage of peptide bonds.

The level of amide nitrogen in the membrane serves as an indicator ofthese changes. For example, it has been found that if the amide nitrogencontent falls by about half (ie. from 0.7 mmol/g to 0.35 mmol/g) thenmore than 95% of the collagen is still present in its native state. Thebasis of this measurement is the hydrolysis of amide groups in the aminoacids asparagine and glutamine: ##STR1##

The degree of purification of the collagen can be determined by aminoacid analysis. Collagen is hydrolysed to form amino acids, which meansthat this analysis indicates pure collagen and elimination ofnon-collagenous proteins but not the denaturing of collagen.

Together with amino acid analysis of the collagen, the glycosamine andproteoglycans content can also be analysed. These contaminants arehydrolysed and the monomeric glycosamine and hexosamine content of themembrane is determined by chromatography. It has been found that thequantity of glycosamine and galactosamine after purification isapproximately 1 molecule to 10,000 molecules of amino acids.

In one method of preparing the membranes, the raw materials are firsttreated with alkali. For this step, solutions of NaOH are used inconcentrations from 0.2-4% by weight. The fats are saponified, and anyaccompanying proteins sensitive to alkalis are eliminated together withany other substances sensitive to alkalis, such as glycosaminoglycans,proteoglycans, etc. The process is controlled by determining the amidenitrogen. At the end of the alkaline treatment the level of amidenitrogen should be between 0.3 and 0.5 mmol/g.

The second step is the treatment of the material with inorganic acid,usually HCl. Acid-sensitive contaminants are eliminated, the fibres aregreatly swollen and in this way the fibrous structure is loosened.Acidification is continued until the material is homogeneouslyacidified.

After this, the material is washed. It has proved useful to wash thematerial until the pH has changed from 0.5-1.5 (during acidificationwith HCl) to between 2.5-3.5. The washing is preferably carried out withdistilled water.

The swollen material can now be levelled out (split), to achieve auniform thickness. Further steps include a de-swelling operation,neutralisation and thorough washing of the material. For this, thematerial is first treated with an acidic (pH 2.8-3.5) common saltsolution (concentration 5-10% by weight). The material is thuscompletely de-swollen. It is then washed with excess of slightlyalkaline distilled water until the pH of the material reaches 5.8-6.5.The material is then thoroughly washed with distilled water (pH 6.0).This brings to an end the first phase of production, namelypurification. This is followed by drying and degreasing.

The material is dried by repeated washing with acetone. This causesshrinkage of the collagen fibres and, as a result, an open structureremains. The degreasing is carried out with n-hexane. This eliminatesthe last traces of hydrophobic substances from the material.

The dry thickness of the membrane for use in guided tissue regenerationaccording to the present invention should ideally be between 0.1 and 1.0mm but can be influenced by swelling of the material.

The membrane may thus be split or sectioned to achieve the requiredthickness, provided that the dual textures of the membrane aremaintained.

The membrane may further be treated to adapt its properties to suit aparticular wound type. Thus, the collagen of the membrane may becross-linked to stabilise the membrane and reduce the rate of absorptionby the body.

All the crosslinking agents known hitherto and used for medical productscan be used for the membranes (e.g. formaldehyde, glutardialdehyde,hexamethylenediisocyanate, dicyclohexylcarbodiimide, etc.). Physically,crosslinking may be carried out by the application of heat. In this casethe crosslinking effect is admittedly smaller but is sufficient for mostapplications. Conveniently the collagen of the membrane is physicallycrosslinked by heating to 100°-120° C. (for 30 minutes to 5 hours),thereby extending the degradation time.

Conveniently the degree of cross-linking introduced will be such thatthe rate of reabsorption of the membrane correlates with the growth ofthe new tissue and healing of the wound. For example, osteocytes takeapproximately 6 weeks to regenerate a tooth cavity and thus a membranewhich is absorbed in a period of 8-12 weeks would be suitable for liningthat wound type. Clearly the membrane should not be heavily cross-linkedotherwise the rate of absorption would be too slow and in extreme casesthe membrane becomes non-absorbable.

One other modification which may be made to the membrane is to coat orimpregnate the fibrous side with a glycosaminoglycan (GAG) such ashyaluronic acid, chondroitin sulphate, dermatan sulphate or keratansulphate.

Glycosaminoglycans such as hyaluronic acid are important as regulatorymolecules which affect tissue structure. They have a favourableinfluence on:

cell infiltration

the formation and degradation of the fibrin matrix

swelling of the matrix

phagocytosis

vascularisation

Shortly after injury the content of GAG in a wound increases. Hyaluronicacid and related GAGs bind to fibrin and form a three dimensional matrix(clot) which is interwoven within the fibrin matrix. The original fibrinmatrix is thereby deformed, swells and becomes more porous. This permitsbetter and faster infiltration and migration of the cells into thematrix.

Hyaluronic acid and fibrinogen react specifically with one another, evenif one or other molecule is in a solid state.

In the inflammatory stage of injury hyaluronic acid stimulatesgranulocyte function, alters the properties of the surface ofpolymorphonuclear leukocytes and regulates the phagocytosis activity ofcells.

During the conversion and breakdown of the hyaluronic acid fibrinmatrix, smaller fragments of hyaluronic acid are produced. Smallfragments of hyaluronic acid stimulate the construction of new bloodvessels.

Additionally, GAGs such a hyaluronic acid make collagen incapable ofprovoking an immune reaction in a host animal. In order to achieve thisthe collagen must be reacted with at least one weight percent of GAGacid.

GAGs are carrier for structural and biologically active proteins. It hasbeen found that GAG protein complex plays a very important part inscar-free wound healing in the fetus.

For these reasons impregnation of the collagen membrane with GAGs suchas hyaluronic acid causes improved tissue regeneration within a wound orbone lesion.

In a further aspect, the present invention provides a membrane for usein guided tissue regeneration, one side of said membrane having a smoothtexture, the opposite side having a fibrous texture, said membrane beingimpregnated with one or more GAGs.

Preferably, the GAG concentration increases through the thickness of themembrane, with the highest concentration of GAG being on the fibrousside of the membrane.

The GAG material may be introduced into the membrane as a gel which isspread onto the fibrous side of the membrane and then allowed to dry.This approach achieves a decreasing concentration gradient down into themembrane whilst the GAG does not completely penetrate through themembrane.

Whilst we do not wish to be bound by theoretical considerations, it isbelieved that the chains of the high MW GAGs act to guide the new cellsdown onto the membrane surface which can then act as a support for cellgrowth.

It is thus particularly beneficial that the fibrous side of the membraneis in the form of a composite matrix including GAGs.

Hyaluronic acid and other GAGs naturally in the body with the skincontaining 19% and the peritoneum 13% (by weight hyaluronic acid. Asnaturally occurring substances GAGs do not cause any problems regardingtoxicity or resorption, but rather are believed to act as a naturalnutritional substance for the cells. Hyaluronic acid and other GAGs areproduced industrially and are thus readily available in commercialquantities.

Conveniently the membrane according to the present invention contains0.1 to 30% by weight of a GAG, for example hyaluronic acid, for example2-10% by weight.

If required other pharmaceuticals such as antibiotics (e.g.tetracycline), chemotherapeutics (e.g. taurolidine) and other drugs mayalso be incorporated into the membrane.

The present invention also provides the use of the resorbable membranedescribed above, optionally including one or more GAGs such ashyaluronic acid as additive in the manufacture of a component matrix foruse as a guided tissue regeneration implant.

One particularly beneficial application of the membranes in guidedtissue regeneration is after orafacial or dental surgery. Here it isoften important for bone regeneration to take place, for example afterpartial removal of a tooth root or section of jaw. The constrictedorofacial area makes surgery difficult and thus a non-toxic fullyresorbable implant for guided tissue regeneration is highlyadvantageous. In addition, the nature of the membrane is particularlysuited to encouraging the growth of osteocytes (bone tissue).

The present invention further provides a method of treating wounds orlesions of the human or non-human animal (preferably mammalian) body,said method comprising application of a membrane as described above tothe wound or lesion, said membrane being orientated so that the fibrousside faces the area where tissue regeneration is required. The method isparticularly suitable for the treatment of orofacial wounds or lesions.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a membrane according to the present invention in use forbone regeneration of a tooth (1) which has suffered heavy bone lossaround the root (2) resulting in a cavity (3) normally filled by healthyroot. The root (2) protrudes through a layer of connective tissue or gum(4) into a bone socket (5). To prevent ingrowth of connective tissue (4)into cavity (3) a membrane cover (6) is placed around the outermost edgeof the wound. The membrane (6) extends all round the wound cavity (3).The membrane (3) has a smooth side (7) which faces away from cavity (3)and a fibrous side (8) which faces into cavity(3). The fibrous side (8)provides a supporting surface for new cells growing outward from root(2), whereas the smooth side (7) of the membrane (6) prevents cells ofconnective tissue (4) invading the wound cavity. Membrane (6) isresorbed slowly back into the body, optimally membrane absorptioncorrelates to the time taken for wound healing.

The present invention may be further illustrated by means of thefollowing non-limiting Example:

EXAMPLE

The peritoneal membranes from young calves are completely freed fromflesh and grease by mechanical means, washed under running water andtreated with 2% NaOH solution for 12 hours. The membranes are thenwashed under running water and acidified with 0.5% HCl. After thematerial has been acidified through its entire thickness (about 3 hours)the material is washed until a pH of 3.5 is obtained. The material isthen shrunk with 7% saline solution, neutralised with 1% NaHCO₃ solutionand washed under running water. The material is then dehydrated withacetone and degreased with N-hexane. The amide nitrogen content of thematerial is 0.47 mMole/g.

Determination of the Amide Nitrogen Content

(Eastoe, E; Courts, A; Practical Analytical Methods for ConnectiveTissue Proteins (1963)).

    ______________________________________                                        Reagents                                                                      1.      2 N HCl (160 ml of conc. HCl made up to 1 litre)                      2.      0.05 M Borax - 0.15 N NaOH solution (19.1 g                                   Na.sub.2 B.sub.4 O.sub.7.10 H.sub.2 O) + 6 g NaOH in 1 litre,                 topped up                                                                     with cold distilled H.sub.2 O.                                        3.      Indicator - mixed in ethanol (0.33% methylene blue +                          0.05% methylene red)                                                  4.      1% boric acid with the indicator                                              10 g boric acid in 1 litre distilled cold H.sub.2 O                           8 ml indicator                                                                0.7 ml 0.1 N - NaOH                                                   5.      0.01 N HCl                                                            Method                                                                        1.      1 g of dried collagen mass is dispersed in 50 ml of                           2 N HCl and boiled for 1 hour. The volume is made                             up to 50 ml at 20° C.                                          2.      5 ml of this dispersion are placed in a                                       microkjehldahl flask, together with 20 ml of                                  solution 2; the micture is distilled in 20 ml of                              solution 4. Distillation takes 6 minutes.                             3.      The solution is titrated with reagent 5.                              Calculation                                                                   ml of acid consumption × 20 = mmol % amide N                            Example                                                                       1.36 ml of 0.01 N HCl × 20 = 27.2 mmol %                                             = 0.27 mmol/g amide N                                            ______________________________________                                    

We claim:
 1. A resorbable collagen membrane for use in guided tissueregeneration comprising a substantially purified collagen membrane whichis physiologically acceptable for implant into a mammalian body, andwhich is at least about 95% by weight native collagen, wherein a firstface of said membrane is fibrous thereby allowing cell growth thereonand an opposite face of said membrane is smooth, thereby inhibiting celladhesion thereon and acts as a barrier to prevent passage of cellstherethrough.
 2. A membrane as claimed in claim 1 which is derived froma natural collagen membrane.
 3. A membrane as claimed in claim 2 derivedfrom mammalian peritoneum or pericardeum, placenta membrane or basalmembrane.
 4. A membrane as claimed in claim 3 which is derived fromcalves or piglets.
 5. A membrane as claimed in claim 4 which is derivedfrom 6-7 week old piglets.
 6. A membrane as claimed in claim 1 which issubstantially free from fat.
 7. A membrane as claimed in claim 1 whichhas a thickness of 0.1 to 1.0 mm when dry.
 8. A membrane as claimed inclaim 1 in which the collagen is cross-linked without becomingnon-resorbable.
 9. A membrane as claimed in claim 1 in which said firstface is on a fibrous side of said membrane, and wherein the fibrous sideis impregnated with a glycosaminoglycan.
 10. A membrane as claimed inclaim 9 in which the fibrous side is impregnated with hyaluronic acid.11. A membrane as claimed in claim 9 in which the fibrous side isimpregnated with a member selected from the group consisting ofchondroitin sulphate, dermatan sulphate, keratan sulphate and mixturesthereof.
 12. A membrane as claimed in claim 9 in which the membranecontains 0.1 to 30.0% by weight of glycosaminoglycan.
 13. A membrane asclaimed in claim 1 containing at least one pharmaceutical.
 14. Amembrane as claimed in claim 13 containing taurolidine.
 15. A method oftreating a wound or lesion of a human or non-human animal bodycomprising application of a membrane as claimed in claim 1 to the woundor lesion, said membrane being so oriented that the fibrous face of themembrane faces an area where tissue regeneration is required.
 16. Amethod as claimed in claim 15 in which the said tissue is bone.
 17. Amethod as claimed in claim 16 in which the wound or lesion is in anorofacial region.
 18. A method of preparing a membrane as claimed inclaim 1 in which a mammalian collagen membrane having a smooth face anda fibrous side having a fibrous face is subjected to treatment withalkali to saponify fats and eliminate alkali sensitive substances andthen acidified to eliminate acid sensitive substances, followed bywashing, drying, and degreasing, whereby to form a membrane comprisingat least about 95% by weight native collagen.
 19. A method as claimed inclaim 18 in which, a glycosaminoglycan is impregnated into the fibrousside of said membrane.
 20. A method of guided tissue regenerationcomprising application of a resorbable collagen membrane to an orofacialregion of a human or non-human animal body, wherein a first face of saidmembrane is fibrous thereby allowing cell growth thereon and an oppositeface of said membrane is smooth, thereby inhibiting cell adhesionthereon and acts as a barrier to prevent passage of cells therethrough.21. A method as claimed in claim 20 wherein said membrane comprises atleast about 95% by weight native collagen.
 22. A method as claimed inclaim 20 in which the tissue is bone.
 23. A method as claimed in claim18 further including the step of cross-linking said membrane.
 24. Amembrane as claimed in claim 12, in which said membrane contains 2-10%by weight said glycosaminoglycan.